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J Biol Chem, Vol. 274, Issue 42, 30222-30227, October 15, 1999
From the Institut für Biochemie, Rheinisch-Westfälische
Technische Hochschule Aachen, 52074 Aachen, Germany,
§ Institut für Medizinische Strahlenkunde und
Zellforschung, Universität Würzburg, 97078 Würzburg,
Germany, and ¶ Medizinische Klinik der Heinrich-Heine
Universität, 40255 Düsseldorf, Germany
Environmental stress (e.g.
aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive
oxygen species activate intracellular signaling cascades such as the
"stress-responsive" mitogen-activated protein kinases and nuclear
factor Mitogen-activated protein
(MAP)1 kinases are important
mediators of signal transduction from the cell surface to the nucleus. Regulation by MAP kinases has been implicated in many cellular processes such as proliferation, differentiation, and apoptosis. In
mammals, MAP kinases are divided into at least three subfamilies: the
"classical" extracellular signal-regulated kinases (extracellular signal-regulated kinases 1 and 2), the stress-activated protein kinases/c-Jun N-terminal kinases (JNK), and the cytokine-suppressive anti-inflammatory drug-binding protein/p38. Whereas extracellular signal-regulated kinase-type MAP kinases are preferentially activated by a variety of cell growth and differentiation stimuli and by hypoosmolarity, JNK and p38 are primarily activated by various environmental stresses (for reviews, see Refs. 1-3). p38 has substantial similarity to the S. cerevisiae HOG1 kinase, a
yeast MAP kinase required for cellular osmoregulation (4). Like HOG1, p38 is activated in response to changes in environmental osmolarity. It
further appears to be involved in the signal transduction of lipopolysaccharide and inflammatory mediators such as tumor necrosis factor Another key signaling system involved in the signal transduction of
numerous interleukins and the interferons as well as a number of growth
and differentiation factors is the Janus kinase (Jak)/signal transducer
and activator of transcription (STAT) pathway. The binding of mediators
to their respective receptors activates tyrosine kinases of the Jak
family, followed by tyrosine phosphorylation, dimerization, and nuclear
translocation of STAT factors (for a review, see Ref. 9). At least upon
IL-6 signaling, also the tyrosine phosphatase SHP2 is subject to
phosphorylation, although its specific function is still unclear. It
has been reported that SHP2 negatively regulates IL-6-induced gene
transcription (10, 11). Further, there is evidence that it acts as an
adaptor linking the Jak/STAT pathway to the MAP kinase pathway via Grb2 (12).
Recently, we have shown that in a number of primary cells and cell
lines, hyperosmotic shock results in the tyrosine phosphorylation of
Jak1, Jak2, and Tyk2 and in the activation of mainly STAT1 and/or
STAT3. Furthermore, an important role of Jak1 in the activation of
STAT by hypertonicity was demonstrated (13).
In this study, we explored the possible interactions between the
Jak/STAT and the p38 kinase pathways under hyperosmotic conditions. We
show that, apart from Jaks and STAT factors, the tyrosine phosphatase SHP2 becomes also phosphorylated upon hyperosmotic shock. We could demonstrate that p38 and its upstream activator MKK6 participate in the
activation of STAT1 and tyrosine phosphorylation of SHP2 by
hyperosmotic shock. Further evidence is given that a protein-tyrosine kinase different from Jak1 is required for hyperosmotic STAT activation.
Materials--
Restriction enzymes were purchased from Roche
Molecular Biochemicals (Mannheim, Germany). Oligonucleotides were
obtained from MWG Biotech (Ebersberg, Germany), and SB 203580 (14) and
SB 202190 (5) were from Calbiochem (Bad Soden, Germany). Dulbecco's modified Eagle's medium (DMEM) was from Life Technologies, Inc. (Eggstein, Germany), and fetal calf serum was from Seromed (Berlin, Germany). Recombinant human IL-6 and soluble IL-6 receptor gp80 were
prepared as described (15, 16). DEAE-dextran and chloroquine were
purchased from Sigma (Deisenhofen, Germany).
Cell Culture and Stimulation of Cells--
COS-7 cells were
grown in DMEM at 5% CO2 in a water-saturated atmosphere.
DMEM was supplemented with 10% fetal calf serum, streptomycin (100 mg/liter), and penicillin (60 mg/liter). Medium was changed and
adjusted to 6 ml 16 h before experiments were carried out.
Cells grown in a 100-mm dish to about 80% confluence were stimulated
with sorbitol by adding 2 ml of 2.4 M sorbitol (final concentration of 600 mM sorbitol) dissolved in cell culture
medium. Controls received the appropriate volume of cell culture
medium. SB 202190 and SB 203580 were dissolved in Me2SO and
added to the culture medium 20-40 min before stimulation at
concentrations as indicated in the figure legends. Nuclear extracts
were prepared as described by Andrews and Faller (17). Protein
concentrations were determined with a Bio-Rad protein assay.
DNA Constructs and Transfection Procedures--
cDNAs for
p38 wild-type and p38(AF) mutant tagged with the FLAG epitope (18) were
cloned into the KRSPA expression vector as described in Flory et
al. (19). PCDNA3-FLAG-MKK6 wild-type and mutants were a kind
gift from Dr. R. Davis (Worcester, MA). Transfection of COS-7 cells was
performed using 10-20 µg of DNA, according to the DEAE-dextran
method described elsewhere (20), with slight modifications. Briefly,
COS-7 cells were grown in 75-cm2 flasks to approximately
90% confluence. 10-20 µg of cDNA were mixed with serum-free
DMEM, and DEAE-dextran and chloroquine were added to a final
concentration of 0.08 mM and 0.4 mg/ml, respectively. Cells
were incubated for 60-80 min at 37 °C under exclusion of gas
exchange and then washed with phosphate-buffered saline, shocked with
10% Me2SO diluted in phosphate-buffered saline for 1 min, and washed again with phosphate-buffered saline. Incubation was continued in DMEM containing 10% serum for at least 16 h; cells were then split and cultured for another 24 h.
Electrophoretic Mobility Shift Assay (EMSA)--
EMSAs were
performed as described previously (21) using a double-stranded
32P-labeled mutated m67SIE oligonucleotide from the
c-fos promotor (m67SIE, 5'-GAT CCG GGA GGG ATT TAC GGG GAA
ATG CTG-3') (22). The protein-DNA complexes were separated on a 4.5%
polyacrylamide gel containing 7.5% glycerol in 0.25-fold TBE (20 mM Tris, 20 mM boric acid, 0.5 mM
EDTA) at 20 V/cm for 4 h. Gels were fixed in 10% methanol, 10%
acetic acid, and 80% water for 1 h, dried, and autoradiographed.
Immunoprecipitation--
Cells were washed twice with
phosphate-buffered saline and solubilized in 1 ml of lysis buffer
(0.5% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 20 mM glycerophosphate, 1 mM
Na3VO4, 0.25 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, and 15% glycerol) for 30 min at
4 °C. Insoluble material was removed by centrifugation, and the cell
lysate was incubated with specific antibodies at 4 °C for a minimum
of 2 h. The immune complexes were bound to protein A-Sepharose (5 mg/ml in lysis buffer) for 1 h at 4 °C. After centrifugation,
the Sepharose beads were washed three times with wash buffer (0.05%
Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 100 mM
NaCl, 1 mM NaF, 1 mM EDTA, 20 mM
glycerophosphate, 1 mM Na3VO4, and
15% glycerol). The samples were boiled in gel electrophoresis sample
buffer, and the precipitated proteins were separated on an
SDS-polyacrylamide (7.5%) gel. The following antibodies were used:
anti-Jak1 rabbit polyclonal antibodies (kindly provided by Dr.
Ziemiecki, Bern, Laboratory for Clinical and Experimental Cancer
Research) and anti-SHP2 rabbit polyclonal antibody from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
Immunoblotting and Immunodetection--
The electrophoretically
separated proteins were transferred onto polyvinylidene difluoride
(PVDF) membranes by the semidry Western blotting method. Nonspecific
binding was blocked with 10% bovine serum albumin in TBS-N (20 mM Tris/HCl, pH 7.4, 137 mM NaCl, and 0.1%
Nonidet P-40) for 15 min. The blots were incubated with primary
antibodies at a 1:1000 dilution in TBS-N for 1 h. After extensive
rinsing with TBS-N, blots were incubated with secondary antibodies,
goat anti-rabbit IgG, or goat anti-mouse IgG conjugated to horseradish
peroxidase for 1 h. After further rinsing in TBS-N, the
immunoblots were developed with the ECL system (Amersham Pharmacia
Biotech) following the manufacturer's instructions. The following
primary antibodies were used: anti-phosphotyrosine mouse monoclonal
antibody (4G10; Upstate Biotechnology, Inc.); anti-Jak1 rabbit
polyclonal antibodies; anti-SHP2 rabbit polyclonal antibody (Santa Cruz
Biotechnology); phosphotyrosine-specific STAT1 (Tyr701)
rabbit polyclonal antibody (New England Biolabs);
phosphoserine-specific STAT1 (Ser727) rabbit polyclonal
antibody (Upstate Biotechnology); anti-p38 rabbit polyclonal antibody
(Santa Cruz Biotechnology); and anti-active p38 rabbit polyclonal
antibody (Promega).
p38 in Vitro Kinase Assay--
Cells were lysed in a modified
lysis buffer (0.5% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 20 mM p38 and Its Upstream Activator MKK6 Are Required for Hyperosmotic
STAT Activation--
Our recent finding that hyperosmolarity leads to
a rapid and ligand-independent activation of the Jak/STAT pathway (13) and the well known fact that an osmotic shock also results in an
activation of the p38 MAP kinase (4) raise the question of whether
these two processes are connected. As shown in Fig. 1, upon hyperosmotic stress, p38 activity
in COS-7 cells as determined by an activation-dependent
antibody and by an in vitro kinase assay using MAP
kinase-activated protein kinase 2 as a substrate parallels activation
of STAT1 assessed by an EMSA with an oligonucleotide from the
c-fos promotor (mutated SIE probe (22)). p38 and STAT remained activated over at least 3 h. We next studied the
influence of SB 202190 or SB 203580 (specific inhibitors of p38) on
hyperosmotic STAT activation. Inhibition of the p38 kinase almost
completely abolished hyperosmotic STAT1 activation as assessed by an
EMSA and anti-phosphotyrosine blotting (Fig.
2). This inhibition was already
detectable at concentrations of 1 µM and maximal at
concentrations higher than 5 µM (Fig. 2A).
Interestingly, IL-6-induced STAT activation in COS-7 cells was not
affected by p38 inhibitors (Fig. 2B, two right lanes). These data suggest that the p38 MAP
kinase might be involved in hyperosmotic STAT activation.
This notion was further substantiated by overexpression of wild type
and kinase-deficient mutants of p38 and of its upstream activator MKK6
in COS-7 cells. In the inactive MKK6(A) mutant, the conserved lysine in
the ATP binding site is replaced by alanine (23), whereas the inactive
p38 kinase was generated by replacement of two activating
phosphorylation sites with alanine and phenylalanine, respectively
(p38(AF)) (18). Transient overexpression of wild-type p38 (Fig.
3A) and MKK6 (Fig.
3B) resulted in an enhanced tyrosine phosphorylation and
binding to a cognate DNA probe of STAT1 upon hyperosmotic stress. In
contrast, overexpression of the dominant-negative mutants p38(AF) (Fig.
4A) and MKK6(A) (Fig.
4B) markedly suppressed the activation of STAT1 by
hyperosmolarity. A faster migrating band below STAT1 is recognized by
both a STAT1- and a phosphotyrosine 701-STAT1-specific antiserum,
suggesting that it might represent STAT1
Since the p38 MAP kinase is a serine/threonine kinase, we asked whether
p38 might affect the serine phosphorylation of STAT1 at serine 727, which lies within a putative MAP kinase consensus motif (25, 26), and
thereby indirectly favor its tyrosine phosphorylation. As depicted in
Fig. 5 phosphorylation at serine 727 of
STAT1 slightly increased under hyperosmotic conditions with a time
course similar to tyrosine phosphorylation. However, overexpression of
wild type or kinase-deficient forms of p38 or MKK6 did not
significantly affect phosphorylation of STAT1 at serine 727. These data
indicate that although activation of the MKK6/p38 pathway is essential
for activation of STAT1 by hyperosmotic shock, it is independent of
phosphorylation at serine 727.
Protein-tyrosine Kinase Activity Independent of Jak1 Is Required
for Hyperosmotic STAT Activation--
The fact that p38 is a
serine/threonine protein kinase necessitates another regulatory step
leading to the tyrosine phosphorylation of STAT1. As shown in Fig.
6A, hyperosmotic STAT
activation is dose-dependently decreased when cells were
treated with genistein, an unspecific inhibitor of protein-tyrosine
kinases, before exposure to hyperosmotic stress. STAT activation was
not affected by equal concentrations of daidzein, the inactive analog
of genistein, demonstrating that the inhibitory effect of genistein is
specific (Fig. 6B). This indicates that a protein-tyrosine
kinase activity is required for activation of STAT1 by hypertonicity.
Studies on Jak1-deficient U4A cell lines suggested that Jak1 is
important for the hyperosmotic STAT activation (13). Therefore, it
appeared likely that the genistein-sensitive tyrosine kinase necessary for hyperosmotic STAT activation is Jak1, possibly regulated by p38.
However, as shown in Fig. 7A,
there was no effect of genistein on tyrosine phosphorylation of Jak1
induced by hyperosmolarity (Fig. 7A). Furthermore, the p38
kinase inhibitors SB 203580 (Fig. 7A) and SB 202190 (Fig.
7B) did not markedly influence stress-induced tyrosine
phosphorylation of Jak1. Since it is currently believed that the
tyrosine phosphorylation of Jak1 is mainly due to autophosphorylation, these data indicate that Jak1 activity is neither responsible for the
genistein-sensitive hyperosmotic STAT activation nor regulated by the
p38 pathway.
Hyperosmotic Activation of the Tyrosine Phosphatase SHP2 Is
Dependent on p38 and Protein-tyrosine Kinase Activity--
STAT
activation could be not only the result of increased tyrosine kinase
activity but also the result of the inhibition of an inactivating
tyrosine phosphatase. SHP2 is a tyrosine phosphatase that has been
connected to the Jak/STAT pathway although its specific function
remains to be elucidated. SHP2 is thought to be a gp130-associated adapter connecting the signal transducer to Grb2, thereby linking the
Jak/STAT to the MAP kinase pathway (12). Further, it has been reported
that SHP2 negatively modulates IL-6-induced gene expression (10, 11).
We therefore asked whether SHP2 plays a role in the hyperosmotic STAT
activation and if so whether it is regulated by the p38 MAP kinase
and/or by genistein-sensitive tyrosine kinases, respectively. Fig.
8 shows that SHP2 becomes tyrosine-phosphorylated within 15 min upon hyperosmotic shock and that
this phosphorylation persisted for at least 45 min. Inhibition of p38
by SB 202190 dose-dependently suppressed also tyrosine phosphorylation of SHP2 (Fig.
9A). In addition,
overexpression of a kinase-deficient mutant of p38 (Fig. 9B)
or MKK6 (Fig. 9C) inhibited tyrosine phosphorylation of
SHP2, whereas overexpression of wild-type kinase had almost no effect.
Inhibition of protein-tyrosine kinases by genistein decreased the
hyperosmotically induced tyrosine phosphorylation of SHP2 (Fig.
9A). Vogel et al. (27) have shown the extent of
tyrosine phosphorylation of SHP2 to correspond to its phosphatase
activity. Considering a regulatory role of SHP2 for the hyperosmotic
STAT activation, phosphorylation of this phosphatase should behave
contrary, not parallel to, STAT activation. Thus, it is most unlikely
that inactivation of the tyrosine phosphatase SHP2 is responsible for
STAT activation upon hyperosmolarity.
Activation of the p38 kinase cascade is known as one of the major
events occurring after hyperosmotic shock (4). Recently, we
demonstrated that also the Jak/STAT pathway is activated by hyperosmolarity. Studies on Jak1-, Jak2-, and Tyk2-deficient cell lines
suggested that Jak1 might be important for the hyperosmotic STAT
activation (13). Here we give evidence that tyrosine phosphorylation and activation of STAT1 in response to hyperosmotic treatment occurs at
least in part via the MKK6/p38 pathway. This was shown by inhibition of
the p38 MAP kinase using specific inhibitors and by overexpression of
wild type or kinase-deficient mutants of p38 and MKK6. Our data further
indicate that the interaction between MKK6/p38 and STAT1 does not
involve Jak1, although protein-tyrosine kinase activity is required for
hyperosmotic STAT activation.
It has been reported that the transactivating capability of STAT1
depends at least partially on the phosphorylation of serine 727, located within a potential MAP kinase consensus motif in the C-terminal
transactivation domain (25, 26, 28, 29). Furthermore, after bacterial
infection or administration of lipopolysaccharide prior to IFN Since p38 is a serine/threonine protein kinase and a direct action of
p38 on STAT1 is unlikely, tyrosine phosphorylation and activation of
STAT1 after hyperosmotic shock must occur indirectly. This could be
either by activation of a tyrosine kinase controlled by
serine-threonine phosphorylation or by inhibition of a tyrosine phosphatase negatively regulated by serine/threonine phosphorylation. A
tyrosine phosphatase that has been frequently associated with the
Jak/STAT pathway is SHP2 (also termed as PTP1D, Syp, or SH-PTP2). The
functional implications of SHP2 for the signal transduction via the
Jak/STAT pathway are largely unknown. It binds with its SH2 domain
specifically to phosphorylated tyrosine 759 from the gp130 receptor
subunit cytoplasmic domain, thereby linking gp130 via Grb2 to the MAP
kinase pathway (12). Further, previous work by Schaper et
al. (10) and Kim et al. (11) showed that activation of
SHP2 limits acute phase protein expression most likely via dephosphorylation of gp130, Jaks, or STATs. Here we show that upon
hyperosmotic shock SHP2 becomes phosphorylated with a time course
similar to STAT1 activation and that this phosphorylation is regulated
at least partially by MKK6/p38 and a genistein-sensitive tyrosine
kinase. It is currently believed that the extent of tyrosine phosphorylation of SHP2 corresponds to its phosphatase activity (27).
Since we did not observe that the SHP2 tyrosine phosphorylation behaves
in reverse to the tyrosine phosphorylation of STAT1, we assume that
inhibition of phosphatase activity of SHP2 is probably not involved in
the activation of STAT1 due to hyperosmotic stress.
Another phosphatase that was reported to negatively regulate STAT
activation is MAP kinase phosphatase 1 (31). This phosphatase belongs
to a class of dual specific phosphatases inactivating extracellular
signal-regulated kinase-type MAP kinases and JNK by dephosphorylation
(32-34). MAP kinase phosphatases are the products of immediate early
genes whose mRNA is rapidly induced following such divergent
stimuli as serum, epidermal growth factor, neural growth factor (32,
35), short wave UV light, and DNA-alkylating agents (34). However, p38
has been suggested to induce transcription of MAP kinase phosphatase 1 upon hyperosmotic stress, since the hyperosmotic induction of MAP
kinase phosphatase 1 mRNA can be blocked by p38 MAP kinase
inhibition (36). Since MAP kinase phosphatase 1 activity is only
regulated on the level of transcription (32), its induction by p38
makes it an unlikely candidate for the hyperosmotic STAT activation via p38.
Another possible mechanism for the MKK6/p38-mediated STAT activation by
hyperosmolarity is via activation of a protein-tyrosine kinase
controlled by serine/threonine phosphorylation by p38. We report here
that genistein, an unspecific inhibitor of protein-tyrosine kinases
inhibits activation of STAT1 and tyrosine phosphorylation of SHP2 upon
hyperosmotic shock, whereas tyrosine phosphorylation of Jak1 is not
affected. This suggests that a genistein-sensitive tyrosine kinase
different from Jak1 is required for hyperosmotic STAT activation and
tyrosine phosphorylation of SHP2. We therefore consider it to be more
likely that a tyrosine kinase, not a tyrosine phosphatase, is the
regulatory step between MKK6/p38 and STAT1 activation upon
hypertonicity. In this respect, it is interesting to note that
depending on the stress employed, activation of JNK can be either the
result of an increased phosphorylation (anisomycin, UV irradiation, and
osmotic stress) or an inhibition of a JNK phosphatase (heat shock,
oxidative stress, and ethanol (37)). If this model of JNK regulation is
also valid for the activation of STAT1 upon hyperosmotic shock, it
would support our notion that a tyrosine kinase is involved.
Identification of this putative tyrosine kinase representing the link
between hyperosmotic STAT activation and p38 will be our future goal.
We thank W. Frisch for technical assistance
and L. Terstegen, S. Thiel, and A. Martens for helpful discussion. We
also thank A. Ziemiecki for kindly providing anti-Jak1 antibodies.
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (DFG) (Bonn), and the Fonds der Chemischen
Industrie (Frankfurt).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
MAP, mitogen-activated protein;
DMEM, Dulbecco's modified Eagle's medium;
dn, dominant negative;
EMSA, electrophoretic mobility shift assay;
IL, interleukin;
Jak, Janus kinase;
JNK, c-Jun N-terminal kinase;
MKK, mitogen-activated protein kinase kinase;
PVDF, polyvinyl difluoride;
PAGE, polyacrylamide gel electrophoresis;
SIE, sis-inducible element;
STAT, signal transducer and activator of transcription;
IB, immunoblot;
IP, immunoprecipitation;
pAb, polyclonal antibody.
The Mitogen-activated Protein (MAP) Kinase p38 and Its Upstream
Activator MAP Kinase Kinase 6 Are Involved in the Activation of Signal
Transducer and Activator of Transcription by Hyperosmolarity*
,
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B. We have recently shown that the Janus tyrosine
kinase/signal transducer and activator of transcription (Jak/STAT)
pathway is ligand-independently activated by hyperosmotic shock. In
the present study, we show that besides STAT1 also the tyrosine
phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity.
SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both
STAT activation and tyrosine phosphorylation of SHP2 induced by
hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated
protein kinase and its upstream activator mitogen-activated protein
kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine
phosphorylation upon osmotic shock. Accordingly, overexpression of
dominant negative mutants of p38 and MKK6 largely decreased
hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2.
Furthermore, we provide evidence that a genistein-sensitive tyrosine
kinase different from Jak1 is involved in stress-activation of STAT1
and tyrosine phosphorylation of SHP2. These results strongly suggest
that hyperosmotic shock activates STAT1 and SHP2 via p38 and its
upstream activator MKK6.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IL-1 (4-6). The major upstream activators of p38 are
the recently discovered dual specific MAP kinase kinases (MKKs) MKK3
and MKK6, while the related MKK4 activates both p38 and JNK (7, 8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 1 mM NaF, 1 mM EDTA, 1 mM
Na3VO4, 5 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 1 mM Pefabloc, and 15%
glycerol), and immunoprecipitation was performed as described above
using p38 rabbit polyclonal antibody (Upstate Biotechnology). The beads
were washed twice with wash buffer (0.05% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 20 mM -glycerophosphate, 1 mM NaF, 1 mM EDTA, 1 mM Na3VO4,
and 15% glycerol) and twice with kinase assay buffer A (20 mM Tris/HCl, pH 7.4, 20 mM
-glycerophosphate, 20 mM MgCl2, and 1 mM Na3VO4). Immunoprecipitates were
mixed with 400 ng of purified MAP kinase-activated protein kinase 2 (Upstate Biotechnology) and 5 µCi of [
-32P]ATP in 20 µl of kinase assay buffer B (20 mM Tris/HCl, pH 7.4, 20 mM -glycerophosphate, 20 mM
MgCl2, 2 mM dithiothreitol, and 1 mM Na3VO4). Incubation was at
30 °C for 30 min. The reaction was terminated by the addition of gel
electrophoresis sample buffer and boiling. The samples were resolved by
10% SDS-PAGE and subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
p38 activity parallels the activation of
STAT1 in COS-7 cells upon hypertonic treatment. COS-7 cells were
treated with 600 mM sorbitol for the times indicated. Cell
lysates were prepared, and after immunoprecipitation with anti-p38
antibodies an in vitro kinase assay with MAP
kinase-activated protein kinase-2 (MAPKAP-K2) as a substrate
was performed as described under "Experimental Procedures"
(third panel). 50 µg of protein from the same
lysate were separated by 10% SDS-PAGE and blotted onto a PVDF
membrane. Membranes were incubated with polyclonal antibodies
specifically raised against the activated form of p38 (top panel). Blots were stripped and reprobed with anti-p38
antibodies (second panel). For the determination
of STAT1 activation, cells were harvested, and nuclear extracts were
prepared and analyzed as described under "Experimental Procedures."
5 µg of nuclear extracts were mixed with a 32P-labeled
oligonucleotide (mutated SIE probe of the c-fos promoter
5'-GAT CCG GGA GGG ATT TAC GGG GAA ATG CTG-3') and EMSAs were
performed. The DNA-protein complexes formed were separated from the
free probe by electrophoresis on a native 4.5% gel (lower panel).
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Fig. 2.
SB 202190 and SB 203580, two specific
inhibitors of the p38 kinase, inhibit hyperosmotic STAT1
activation. Following a preincubation period for 30 min
(A) with SB 203580 or 40 min (B) with SB 202190 at the concentrations indicated, COS-7 cells were treated with 600 mM sorbitol or 200 units/ml IL-6 plus 0.5 µg/ml soluble
interleukin-6 receptor gp80 (sgp80) for 20 min. Cells were
harvested, and nuclear extracts were prepared. EMSAs were performed as
described in the legend to Fig. 1 (lower panel).
For immunoblots, 40 µg of proteins were separated by 7.5% SDS-PAGE.
Immunoblots were developed using specific antibodies directed against
STAT1 phosphorylated at tyrosine 701 (top panel)
and as loading control reprobed with antibodies specific for STAT1
(second panel).
(former p84 (24)). However,
although this protein is apparently constitutively phosphorylated, it
does not bind to the SIE probe in an EMSA. Therefore, it could also
represent an unrelated polypeptide that is unspecifically recognized by
the antisera.
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Fig. 3.
STAT factor activation after hyperosmotic
shock is dependent on p38 kinase and its upstream activator MKK6.
A, COS-7 cells were transfected with either the empty KRSPA
(control) vector or KRSPA-FLAG-p38 wild type (p38
wild-type). B, COS-7 cells were transfected with either
the empty pcDNA3 (control) vector or with the pcDNA3-FLAG-MKK6
wild type (MKK6 wild-type). After 2 days, cells were treated
with 600 mM sorbitol for the times indicated, after which
cells were harvested, and nuclear extracts were prepared as described
in the legend to Fig. 1. For immunoblots, 40 µg of proteins were
separated by 7.5% SDS-PAGE. Immunoblots were developed using specific
antibodies directed against STAT1 phosphorylated at tyrosine 701 (top panel) and as loading control reprobed with
antibodies specific for STAT1 (second panel). 5 µg of nuclear extracts were mixed with a 32P-labeled
oligonucleotide (mutated SIE probe of the c-fos promoter),
and EMSAs were performed (lower panel).
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Fig. 4.
Overexpression of dominant negative mutants
of p38 and MKK6 largely inhibits hyperosmotic activation of STAT1.
A, COS-7 cells were transfected with either the empty KRSPA
(Control) vector or with a FLAG-tagged kinase-deficient
mutant of p38 (AF) (p38 dn). B, COS-7 cells were
transfected with either the empty pcDNA3 (control) vector or with a
FLAG-tagged kinase-deficient mutant of MKK6 (A) (MKK6 dn).
After 2 days, cells were treated with 600 mM sorbitol for
the times indicated, after which cells were harvested, and nuclear
extracts were prepared as described in the legend to Fig. 1. For
immunoblots, 40 µg of proteins were separated by 7.5% SDS-PAGE.
Immunoblots were developed using specific antibodies directed against
STAT1 phosphorylated at tyrosine 701 (top panel) and as loading control reprobed with antibodies
specific for STAT1 (second panel). 5 µg of
nuclear extracts were mixed with a 32P-labeled
oligonucleotide (mutated SIE probe of the c-fos promoter),
and EMSAs were performed (lower panel).
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Fig. 5.
Serine phosphorylation of STAT1 at serine 727 slightly increased after hyperosmotic stress but was not significantly
altered by overexpression of wild-type or kinase-deficient mutants of
p38 or MKK6. In order to determine phosphorylation of STAT1 at
serine-727 upon hyperosmotic shock, immunoblots with nuclear extracts
from the experiments described in the legends to Figs. 4 and 5 were
analyzed using antibodies specific for STAT1 phosphorylated at serine
727. COS-7 cells were transfected with either the empty KRSPA
(Control) vector or with wild-type (p38
wild-type, upper panel) or kinase-deficient
mutant of p38 (p38 dn, third panel).
For the second and the lower panel,
COS-7 cells were transfected with either the empty pcDNA3
(Control) vector or with wild-type (MKK6
wild-type, second panel) or kinase-deficient
mutant of MKK6 (MKK6 dn, lower panel).
After 2 days, cells were treated with 600 mM sorbitol for
the times indicated, after which cells were harvested, and nuclear
extracts were prepared as described in the legend to Fig. 1. 40 µg of
proteins were separated by 7.5% SDS-PAGE and blotted onto a PVDF
membrane. For loading control, see Figs. 4 and 5.
View larger version (37K):
[in a new window]
Fig. 6.
Hyperosmotic activation of STAT1 is inhibited
by genistein. After a preincubation period of 30 min with
genistein (A and B) or daidzein (B) at
the concentrations indicated, COS-7 cells were treated with 600 mM sorbitol for 20 min. Cells were harvested, and nuclear
extracts were prepared as described in the legend to Fig. 1. 5 µg of
nuclear extracts were mixed with a 32P-labeled
oligonucleotide (mutated SIE probe of the c-fos promoter),
and EMSAs were performed.
View larger version (31K):
[in a new window]
Fig. 7.
Inhibition of p38 MAP kinase or
protein-tyrosine kinases does not markedly influence stress-induced
tyrosine phosphorylation of Jak1. Following a preincubation period
of 30 min with genistein (100 µM), daidzein (100 µM), or SB 203580 (20 µM) (A)
and 40 min with SB 202190 (20 µM) (B), COS-7
cells were treated with 600 mM sorbitol for 20 min. Cell
lysates were prepared, and immunoprecipitation with anti-Jak1 antibody
was performed as described under "Experimental Procedures."
Precipitated proteins were separated by 7.5% SDS-PAGE, blotted onto a
PVDF membrane, and analyzed with a specific anti-phosphotyrosine
antibody (upper panel). Blots were stripped and
reprobed with anti-Jak1 antibody to verify equal loading
(lower panel).
View larger version (24K):
[in a new window]
Fig. 8.
SHP2 becomes tyrosine phosphorylated upon
hyperosmotic shock. COS-7 cells were treated with 600 mM sorbitol for the times indicated. Cell lysates were
prepared, and immunoprecipitation with anti-SHP2 antibody was performed
as described under "Experimental Procedures." Precipitated proteins
were separated by 7.5% SDS-PAGE, blotted onto a PVDF membrane, and
analyzed with a specific anti-phosphotyrosine antibody
(upper panel). Blots were stripped and reprobed
with anti-SHP2 antibody to verify equal loading (lower panel).
View larger version (46K):
[in a new window]
Fig. 9.
Stress induced tyrosine phosphorylation of
SHP2 depends on p38 and MKK6 activity. A, after a
40-min pretreatment with SB 202190, genistein, or daidzein at the
concentration indicated, COS-7 cells where treated with 600 mM sorbitol. B, COS-7 cells were transfected
with either the empty KRSPA (Control) vector or with wild
type (p38wt) or the kinase-deficient mutant of p38
(p38dn). After 2 days, cells were treated with 600 mM sorbitol for 20 min. C, COS-7 cells were
transfected with either the empty pcDNA3 (Control)
vector or with wild type (MKK6 wt) or with the
kinase-deficient mutant MKK6 A (MKK6 dn). Cell lysates were
prepared, and immunoprecipitation with anti-SHP2 antibody was performed
as described under "Experimental Procedures." Precipitated proteins
were separated by 7.5% SDS-PAGE, blotted onto a PVDF membrane, and
analyzed with a specific anti-phosphotyrosine antibody
(upper panels). Blots were reprobed with
anti-SHP2 antibody to verify equal loading (lower panels).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
treatment, phosphorylation of STAT1 at tyrosine 701 and at serine 727 occurred independently from each other, leading to an increased
transcriptional response (30). However, tyrosine-phosphorylated STAT1
was found to a larger extent to be also phosphorylated on serine 727 as
compared with non-tyrosine-phosphorylated STAT1, indicating that serine
phosphorylation of STAT1 possibly facilitates tyrosine phosphorylation
(30). Thus, one could speculate that p38 modulates tyrosine
phosphorylation of STAT1 by phosphorylation of serine 727. However,
another group recently presented in vitro and in
vivo evidence that the extracellular signal-regulated kinase-type MAP kinases specifically phosphorylate STAT3 at serine 727 in response
to growth factors negatively modulating STAT3 tyrosine phosphorylation
(26). Moreover, these authors could show that neither JNK1 nor p38 was
able to phosphorylate STAT1 or STAT3 in vitro when
immunoprecipitated from cell lysates prepared after stimulation with
600 mM sorbitol (26). In correspondence to these
observations, we found in this study that overexpression of wild type
or kinase-deficient forms of p38 or MKK6 did not significantly affect
phosphorylation of STAT1 at serine 727. Therefore, our data support the
assumption that STAT1 is not a substrate of p38 in vivo
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of a DFG fellowship.
To whom correspondence should be addressed: Institut für
Biochemie, Klinikum der RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, Germany. Tel.: 49-241-80-88-830; Fax: 49-241-88-88-428; E-mail: Heinrich@RWTH-Aachen.de.
ABBREVIATIONS
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